This invention relates to nucleic acid and amino acid sequences of two delta-6 desaturase homologs and to the use of these sequences in the diagnosis, treatment, and prevention of cardiovascular diseases, disorders of aging, disorders of fatty acid metabolism, and cancer.
Fatty acid desaturases catalyze the elongation and desaturation of the physiologically important hydrocarbon chain molecules known as fatty acids. Fatty acids are lipophilic modifiers of proteins, fuel molecules, insulating materials, and precursors to hormones including the prostaglandins, which are active in pyrogenesis, vasodilation, vasoconstriction, and pain reception. Phospholipids and glycolipids that compose cell membranes are made from fatty acids. Mammals derive unsaturated fatty acids from the unsaturated 16-carbon palmitate or diet-derived linoleate and linolenate, which are desaturated at two and three points, respectively. In eukaryotes, a variety of unsaturated fatty acids can be formed from oleate by a combination of elongation and desaturation reactions occurring on the cystolic face of the endoplasmic reticulum membrane. Desaturation reactions require NADH and O2 and are carried out by a complex consisting of a flavoprotein, a cytochrome, and a nonheme iron protein. The three components of the desaturase complex co-extract under pressure. It has been noted that each of the components of the delta-6 desaturase complex physically face a similar lipid environment in the microsomal membrane. (Leikin et al. (1994) Biochim. Biophys. Acta 1211:150-155.)
It is suggested that interaction between histidine-rich motifs in oleate desaturase could contribute to an iron binding site in the cytoplasmic domain of the protein that would facilitate interaction between the desaturase and cytochrome b5, the source of electrons for the desaturation reaction. (Okuley et al.(1994) Plant Cell 6:147-158.) Zinc also appears to play a role in delta-6 desaturation. (Eder, K. et al. (1996) Biol. Trace Elem. Res.54: 173-183.) A variety of delta-6 desaturase inhibitors are known, e.g., anti-oxidants and calcium channel blockers, and delta-6 desaturase activity in the body is regulated by several hormones. Inhibitors include, e.g., glucagon, epinephrine, ACTH, and glucocorticoids, while insulin and thyroxin are necessary to delta-6 desaturase activity, and growth hormone appears to enhance delta-6 desaturase activity. (Nakamura, M. T. et al. (1996) Lipids 31(2) 139-143.) Expression of desaturase genes may also be influenced by diet. (Kaestner et al. (1989) J. Biol. Chem. 264:14755-14761.)
Several desaturases have been identified, including more than one delta-6 desaturase. (Marzo, I. et al.(1996) Biochim. Biophys. Acta 1301:263-272.) The presence of three histidine boxes and the HX(2-3)(XH)H amino acid motif are used to characterize membrane-bound fatty acid desaturases. (Schmidt et al. (1994) Plant Mol. Biol. 26:631-642; Mitchell et al. (1997) J. Biol. Chem. 272 (45):28281-28288.) A cytochrome b5 domain may also be used to characterize desaturases. A cDNA encoding the delta-6 desaturase from Borago officinalis that contained an N-terminal domain related to cytochrome b5 has been isolated. (Sayanova, O. et al. (1997) Proc.Natl. Acad. Sci.USA,94: 4211-4216.)
Delta-6 desaturase is involved in a variety of diseases and disorders associated with fatty acid metabolism. A decrease in delta-6 desaturase activity is present in rats subjected to long-term exposure to ethanol. Diabetes mellitus involves many of the hormones that regulate delta-6 desaturase activity, such as insulin and glucagon. Obesity has been related to changes in delta-6 desaturase activity, and reduced delta-6 desaturase activity has also been implicated in coronary diseases. Delta-6 desaturation appears to decline with age, especially in women. Cancer cells show decreased delta-6 desaturase activity. Administration of delta-6 desaturation product, gamma linolenic acid, has been shown to ameliorate disorders of metabolism such as coronary disease, diabetes, osteoporosis, and ulcerative colitis, as well as disorders of cell proliferation such as tumorigenesis.
The discovery of a new delta-6 desaturase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of cardiovascular diseases, disorders of aging, disorders of fatty acid metabolism, and cancer.
The invention features substantially purified polypeptides, delta-6 desaturase homologs, referred to collectively as xe2x80x9cD6DHxe2x80x9d and individually as xe2x80x9cD6DH-1xe2x80x9d and xe2x80x9cD6DH-2.xe2x80x9d In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 or SEQ ID NO:3, or to a fragment of either of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3, as well as a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder of aging, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a cardiovascular disease, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a disorder of fatty acid metabolism, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.